Abstract A fundamental question in biology is how cells integrate information from extra- and intra- cellular stimuli to make cell fate decisions. One of the most critical is the cellular choice between proliferation and quiescence. Correct execution of the proliferation-quiescence decision is important in many biological settings, from developmental processes to adult tissue homeostasis, and dysregulation of this decision point occurs in nearly all types of cancer. Yet despite obvious medical relevance, we have a poor understanding of the inputs that control the choice between proliferation and quiescence, and when in the cell cycle cells integrate presence or absence of these inputs. This proposal aims to revisit fundamental questions about the requirements for cellular proliferation using new single-cell technology that allows us to measure multiple nodes simultaneously in living cells. The current paradigm, based on cells released from serum starvation-induced quiescence, is that mitogen and amino acid sensing occurs in G1 phase of the cell cycle prior to the Restriction Point. However, new data suggest that the classic Restriction Point model pertains only to cells coming out of quiescence, and that in actively cycling cells, mitogen, amino acid, and stress signaling impinges on G2 phase of the preceding cell cycle, to dictate the proliferation-quiescence decision immediately after mitosis. Testing this idea requires performing longitudinal time-lapse studies tracking signaling events in thousands of single cells in combination with acute perturbations to establish cause and effect relationships. The goal of this proposal is to develop a combined experimental-computational pipeline involving enzyme activity sensors, CRISPR-based fluorescent protein tagging, high- throughput long-term time-lapse microscopy, single-cell tracking, and big data analysis, toward the goal of developing a dynamic view of signaling inputs that govern proliferation. These technological advances will lead to fundamental knowledge about cell-cycle regulation, while also potentially revealing medically relevant interventions for controlling the proliferation- quiescence decision in cancer.